Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.     

Unfolding of ubiquitin studied by picosecond time-resolved fluorescence of the tyrosine residue

The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25 degrees C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and excited-state tyrosinate. Second, at pH 1.5, the water-exposed tyrosine of either thermally or chemically unfolded UBQ decays as a sum of two exponentials. The double-exponential decays were interpreted and analyzed in terms of excited-state intramolecular electron transfer from the phenol to the amide moiety, occurring in one of the three rotamers of tyrosine in UBQ. The values of the rate constants indicate the presence of different unfolded states and an increase in the mobility of the tyrosine residue during unfolding. Finally, from the pre-exponential coefficients of the fluorescence decays, the unfolding equilibrium constants (KU) were calculated, as a function of temperature or denaturant concentration. Despite the presence of different unfolded states, both thermal and chemical unfolding data of UBQ could be fitted to a two-state model. The thermodynamic parameters Tm = 54.6 degrees C, DeltaHTm = 56.5 kcal/mol, and DeltaCp = 890 cal/mol//K, were determined from the unfolding equilibrium constants calculated accordingly, and compared to values obtained by differential scanning calorimetry also under the assumption of a two-state transition, Tm = 57.0 degrees C, DeltaHm= 51.4 kcal/mol, and DeltaCp = 730 cal/mol//K.     

Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.

The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.     

Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.     

Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: studies of ribonuclease

Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677-4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies--a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.     

Structure of stable protein folding intermediates by equilibrium phi-analysis: the apoflavodoxin thermal intermediate

Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and fluorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-defined states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-defined states.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Study of multiple unfolding trajectories and unfolded states of the protein GB1 under the physical property space

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimensional property space was reduced to a 3-dimensional principle component property space. Under the property space, the multiple unfolding trajectories look like "trees", which have some common characters. The "root of the tree" corresponds to the native state, the "bole" homologizes the partially unfolded conformations, and the "crown" is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight "bole" and "crown" corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of "the standstill in the middle tree bole", which may correspond to a three-state unfolding pathway. The third one has the character of "the circuitous bole" corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.     

Abstracts: Albany 2007, The 15th Conversation

Abstract

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimentional property space was reduced to a 3-dimentional principle component property space. Under the property space, the multiple unfolding trajectories look like “trees”, which have some common characters. The “root of the tree” corresponds to the native state, the “bole” homologizes the partially unfolded conformations, and the “crown” is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight “bole” and “crown” corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of “the standstill in the middle tree bole”, which may correspond to a three-state unfolding pathway. The third one has the character of “the circuitous bole” corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.     

The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr

Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.     

Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.